Bs 13496r

The tissue microarray chip and slides were deparaffinized and rehydrated using xylene and a graded series of alcohols. Endogenous peroxidases were removed through incubation with 3% H₂O₂ for 20 min at 37 °C. Then, slides were autoclaved in 10 mM sodium citrate (pH 6.0) for 30 min to unmask antigens, washed in TBS (pH 7.4) three times, and incubated with primary antibodies against GPAA1 (1:300, Bioss, bs-13496R), ERBB2 (1:300, abcam, ab16901), p-AKT (1:100, Cell Signaling Technology (CST), #4060), Ki-67 (1:400, Bioss, bs-23105R), MMP2 (1:300, abcam, ab97779), MMP9 (1300, abcam, ab38898), the urokinase receptor (UPAR) (1500, abcam, ab218106) at 4 °C overnight. Slides were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, and signal amplification and detection were conducted using DAB according to the manufacturer’s instructions (CST). Finally, images were acquired with a Nikon microscope.

Cells lysis was performed with total protein extraction buffer (NCM Biotech, Suzhou, China), and tissues were homogenized in T-PER tissue protein extraction reagent (Thermo Fisher Scientific), to which a combination of protease and phosphatase inhibitors was added (NCM Biotech, Suzhou, China). The protein concentration was measured by a BCA Protein Assay Kit (Thermo Fisher Scientific). Cell or tissue lysates (approximately 20 μg of protein) were subjected to SDS-PAGE (8–10%) and transferred to nitrocellulose (NC) membranes. After blocking with 5% fat-free milk, the NC membranes were incubated with different primary antibodies at 4 °C overnight. Antibodies against the following proteins were used: GPAA1 (1:1000, Bioss, bs-13496R), caveolin-1 (1:1000, Abcam, ab2910), AP2B1 (1:1000, Proteintech, 15,690–1-AP), β-actin (1:10000, Abcam, ab5644), EGFR (1:1000, Abcam, ab52894), p-EGFR (Y1068) (1:1000, Abcam, ab40815); ERBB2 (1:1000, Abcam, ab16901); p-ERBB2 (Y877) (1:1000, Abcam, ab47262), p-AKT (S473) (1:1000, CST, #4060), and AKT (1:1000, CST, #4685). After three washes in TBST (pH 7.4), membranes were incubated with species-specific secondary antibodies (Thermo Fisher Scientific) at room temperature for 1 h. Ultimately, protein bands were visualized by an Odyssey imaging system (LI-COR Biosciences, Lincoln, NE, USA).